The study of RNA is critical for applications in basic biology and in the diagnosis and treatment of disease. Recently, researchers have determined that the translation of many mRNA sequences relies not only on proper quantities of mRNA expression, but also the active transport of transcripts to subcellular compartments where highly localized translation can occur [Jansen, Nat Rev Mol Cell Biol 2: 247 (2001)]. For example, Beta-actin localizes at the leading lamellae of growing fibroblasts, driving cell motility [Oleynikov et al., Current Biology 13: 199 (2003)]. Unfortunately, despite the importance of these two aspects in mRNA function, there is no tool available to both measure intracellular concentration and observe localization of mRNA in live cells. The NanoFlare (NF) architecture, a Spherical Nucleic Acid (SNA) construct capable of determining relative mRNA concentration levels in live cells has previously been described [Seferos et al., Journal of the American Chemical Society 129; 15477 (2007); Prigodich et al., Analytical Chemistry 84: 2062 (2012); Rosi et al., Science 312: 1027 (2006); Prigodich et al., ACS Nano 3, 2147 (2009)].
The study of RNA is a critical component of biological research and in the diagnosis and treatment of disease. Recently, the localization of mRNA has emerged as an essential process for a number of cellular processes, including restricting certain proteins to specific compartments within cells [Thomas et al., Cell. Mol. Life Sci. 71: 2219 (2014)]. For instance, synaptic potentiation, the basis of learning and memory, relies upon the local translation of specific mRNAs in pre- and post-synaptic compartments [Weiler et al., Proceedings of the National Academy of Sciences 94: 5395 (1997)]. Likewise, the misregulation of RNA distribution is associated with many disorders, ranging from mental retardation and autism to cancer metastasis [Liu-Yesucevitz et al., The Journal of Neuroscience 31: 16086 (2011); Bassell et al., Neuron 60, 201 (2008); Shestakova, E. A.; Wyckoff, J.; Jones, J.; Singer, R. H.; Condeelis, J. Cancer Research 1999, 59, 1202]. However, despite the significant role of mRNA transport and localization in cellular function, the available methods to visualize these phenomena are severely limited. For example, Fluorescence In Situ Hybridization (FISH), the most commonly used technique to analyze spatial distribution of RNA, requires fixation and permeabilization of cells prior to analysis. As a result, analysis of dynamic RNA distribution is restricted to a single snapshot in time. With such a limitation, understanding the translocation of RNA with respect to time, cell cycle, or external stimulus is difficult or impossible. Further, fixed cell analysis is a highly specialized procedure, due to the number steps necessary to prepare a sample. Fixation, permeabilization, blocking, and staining processes each require optimization and vary based on cell type and treatment conditions, rendering FISH prohibitively complicated in many cases. Likewise, live cell analysis platforms such as molecular beacons require harmful transfection techniques such as microinjection or lipid transfection, and are rapidly sequestered to the nucleus upon cellular entry. Thus, in order to accurately study the dynamics of intracellular RNA, a new type of analysis platform is required.